Defects in the biosynthesis of thyroid hormones have been found in some patients with benign and malignant tumors of the thyroid. However, a systematic and precise definition of these biochemical defects has not been possible because of a lack of adequate knowledge concerning this fundamental biosynthetic mechanism. Hence, our objective is to understand the molecular events involved in the biosynthesis of thyroid hormones, a process which is catalyzed by the integral membrane enzyme, thyroid peroxidase (TPO). In this application, we propose: (1) to study the catalytic, regulatory, and structural properties of TPO isolated from porcine thyroid membranes, and (2) to apply our biochemical findings to studies of normal and abnormal human thyroid tissue. We plan to accomplish this by focusing our biochemical studies on the membrane nature and function of TPO and on the mechanism of TPO-catalyzed iodination. Specifically, we propose: (a) to determine what facets of TPO-catalyzed iodination result from the integral membrane location of TPO by comparing the kinetic and structural properties of intact and fragmented TPO, and (b) to define the TPO-associated iodinating intermediate by characterizing the chemical nature of its active site and its interaction with halogen acceptors, including the thiocarbamide antithyroid drugs. In order to correlate the biochemical findings from our studies on porcine TPO with clinical studies on defects in thyroid hormone biosynthesis, we plan to study the biochemical properties of TPO in normal and abnormal human thyroid tissue, with particular emphasis on the membrane associated nature of human TPO; to investigate the feasibility of using thyroid needle-biopsy samples for a TPO solubilization test which may help to differentiate between benign and malignant thyroid tissue; and to investigate the unusual hydrophobic properties of TPO in human thyroid cancer membranes.